Synthesis and application of procainamide analogs for use in an immunoassay

ABSTRACT

The present invention relates to immobilized procainamide analogs, as well as to a method of making immobilized procainamide analogs. These immobilized analogs are prepared by activating the carboxyl group on a substituted p-benzoic acid derivative toward nucleophilic attack; reacting the activated benzoic acid derivative with a polyamine to produce the benzoic acid derivative of Formula 3: 
                         
and binding the benzoic acid derivative of Formula 3 to a latex polymer having functional groups that react with aliphatic amino groups. A method of conducting an immunoassay using the immobilized procainamide analog Formula 3 is described, comprising the steps of preparing a solution comprising said immobilized procainamide analog; adding a sample suspected of containing procainamide to said solution; adding an anti-procainamide antibody to said solution and observing the rate of increase in solution turbidity following antibody addition.

FIELD OF THE INVENTION

The present invention relates to novel derivatives of procainamide. Thederivatives may be used as immunogens to stimulate antibody production,or to produce particulate polymer conjugates useful in immunoassays fordetection of procainamide. Also provided are methods for synthesis ofthe procainamide derivatives and their polymer conjugates.

BACKGROUND OF THE INVENTION

The ability to assess whether an individual has been exposed to apharmacological agent, and a capability of determining the concentrationof such an agent in a biological sample is of broad importance inmedicine, law enforcement and other areas. In particular, the narrowwindow of therapeutic utility, and the proximity of that window totoxicity for many drugs such as Procainamide have necessitated thedevelopment of assays capable of detecting and quantifying suchsubstances.

Procainamide is a pharmaceutical agent having utility in treatment ofirregular heartbeats. It functions to restore the heartbeat to a normalrhythm and to slow an overactive heart, thereby allowing the heart towork more efficiently. Procainamide produces its beneficial effects byslowing nerve impulses in the heart and reducing sensitivity of hearttissues. It is important to monitor procainamide levels to ensure that apatient is receiving the correct dose. This is particularly importantwith procainamide, as monitoring helps avoid such side effects ofprocainamide overdose as fast and irregular heartbeat, confusion,stupor, decreased blood pressure, fainting, and cardiac arrest. Areliable test for procainamide in the blood also is useful for assistingmedical personnel in the diagnosis of the cause of less commonprocainamide-induced neurological side effects such as hallucinations,depression, and psychosis.

Immunoassays are assay systems that exploit the ability of an antibodyto specifically recognize and bind to a particular target molecule, andoperate by incubating an antibody that is capable of binding to apredetermined analyte molecule with a sample that is suspected ofcontaining analyte. The concentration of the target molecule isproportional to the concentration of antibody-analyte immune complexes.In some immunoassays, the antibody is bound to a support. Free targetmolecule is allowed to react with the support, and the concentration ofthe target molecule is determined by measuring the concentration ofantibody-analyte complex immobilized to the support.

Target molecules that have become bound to the immobilized antibody canbe detected through the use of a labeled, second antibody that iscapable of binding to a second binding site on the target molecule(i.e., a “sandwich” immunoassay). Immobilization of the labeled antibodyon the support is proportional to the concentration of the target in thesample. Alternatively, in a competitive assay, the sample is incubatedwith a known amount of labeled target and an immobilized antibody. Thetarget molecules in the sample compete with the labeled target moleculesfor the antibody binding sites. Thus, the concentration ofantibody-bound labeled target molecules is inversely proportional to theconcentration of target molecule in the sample.

The utility of an immunoassay in detecting an analyte depends upon itscapacity to report the extent of the formation of immune complexesbetween the antibody employed and the analyte whose presence orconcentration is being measured. In general, two independent approachesexist for increasing this capacity. The first approach involves labelingone or more of the reagents.

Another approach involves increasing the size of the immune complex tothe point where it becomes capable of scattering light. In such cases,agglutination or turbidimetric immunoassay methods may be employed.Turbidimetric methods measure the reduction of light transmitted throughthe suspension of particles or aggregates. The reduction is caused byreflection, scatter, and absorption of the light by the aggregates. Inturbidimetric assays, the rate of change in light scatter may also bemeasured, and provides an indication of the amount of antigen present.

Turbidimetric assays for therapeutic drugs and drugs of abuse which usehapten coated particles are commercially available. An example of suchan assay is a particle-enhanced turbidimetric-inhibition immunoassay(PETINIA). This immunoassay format uses drug-hydrophilic linker-particlereporter reagents (particle reagents). These particle reagents utilizevery small latex particles (e.g., 70 nm) to which have been attached adrug or other compound of interest. When viewed with monochromatic lighthaving a wavelength (e.g., 340 nm) larger than the diameter of thesuspended particles, e.g., the suspension is relatively transparent.Under optimal conditions, addition of antibody specific for the drug onthe particles will cause the particles to agglutinate, forming insolublecomplexes. These complexes cause the suspension to become turbid andscatter light. When an antibody is added to a sample containingdrug/particle conjugates and free drug, free drug competes withparticle-bound drug for antibody, thereby inhibiting both the rate andextent of agglutination. This provides the basis for quantifying theamount of drug in the sample. Specifically, in the present invention,procainamide analog-hydrophilic spacer-particle reagents andanti-procainamide antibodies enable a rapid, precise and accuratePETINIA method for determining the amount of procainamide present inbiological fluids. Such methods, by enhancing both the rate of immunecomplex formation, and the size of the immune complex, provide moreefficient and effective immunoassays for determining the concentrationof medically important pharmacological agents, such as procainamide. Thepresent invention provides reagents and methods for conducting suchimproved immunoassays.

It is an object of the invention to provide novel procainamide analogshaving a linking group that may be used to immobilize them to a support,in particular polymeric support. It is another object of the inventionto provide novel methods of synthesizing biologically activeprocainamide analogs from aminobenzoic acids. It is also an object ofthe invention to provide particulate procainamide analog/polymerconjugates that may be used to detect the presence of procainamideantibodies. It is a further object of the invention to provide animmunoassay for procainamide which functions by measuring changes in theturbidity of a solution or dispersion containing a known quantity of aparticulate procainamide analog/polymer conjugate, a known quantity of aprocainamide antibody, and a sample suspected to contain procainamide.

The foregoing objects and advantages of the invention are illustrativeof those that can be achieved by the present invention and are notintended to be exhaustive or limiting of the possible advantages whichcan be realized. Thus, these and other objects and advantages of theinvention will be apparent from the description herein or can be learnedfrom practicing the invention, both as embodied herein or as modified inview of any variation which may be apparent to those skilled in the art.Accordingly, the present invention resides in the novel methods,arrangements, combinations and improvements herein shown and described.

SUMMARY OF THE INVENTION

In light of the present need for procainamide-based assays, a briefsummary of the present invention is presented. Some simplifications andomission may be made in the following summary, which is intended tohighlight and introduce some aspects of the present invention, but notto limit its scope. Detailed descriptions of a preferred exemplaryembodiment adequate to allow those of ordinary skill in the art to makeand use the invention concepts will follow in later sections.

In one embodiment, the present invention relates to procainamide analogsof Formula 3:

where each P is a protecting group, an alkyl group, an acyl group, or ahydrogen atom; R is hydrogen or lower alkyl; and n is 1, 2, or 3. Theinvention further relates to a method of making procainamide analogs byactivating the carboxyl group on a substituted benzoic acid derivativeof Formula 1:

toward nucleophilic attack. The activated benzoic acid derivative isthen reacted with a polyamine of Formula 2,

to produce a benzoic acid derivative of Formula 3. The analogues ofFormula 3 may then be reacted with a compound of Formula 4:

where X is a leaving group; each CY₂ group is a methylene group, analkylmethylene group, or a carbonyl group; L is an alkylene group havingfrom 2 to 10 carbons or a group of the Formula (CH₂CH₂O)_(p)CH₂CH₂,where p is between 1 and 4; and P₂ is a protecting group. The result isan analog of Formula 5:

In another embodiment, the present invention relates to immobilizedprocainamide analogs, as well as to a method of making immobilizedprocainamide analogs. These immobilized analogs are prepared byactivating the carboxyl group on the substituted benzoic acid derivativeof Formula 1 toward nucleophilic attack; reacting the activated benzoicacid derivative with the polyamine of Formula 2 to produce the benzoicacid derivative of Formula 3; and binding the benzoic acid derivative ofFormula 3 to a latex polymer having functional groups that react withaliphatic amino groups, producing an immobilized procainamide analog ofFormula 3a:

where polymer bound to the procainamide moiety by the wavy line is asupport; in a particular embodiment, a latex polymer particle, which isbound either directly or indirectly to procainamide.

In a third embodiment, a method of conducting an immunoassay using theimmobilized procainamide analogue Formula 3a is described, comprisingthe steps of preparing a solution comprising said immobilizedprocainamide analog; adding a sample suspected of containingprocainamide to said solution; adding an anti-procainamide antibody tosaid solution; and monitoring the rate of the increase in the solutionsturbidity. Further, the method may include a step of observing anychanges in solution turbidity following sample addition to rule outsample-induced agglutination.

DESCRIPTION OF THE DRAWINGS

In order to better understand the present invention, reference is madeto the accompanying drawings, wherein:

FIG. 1 shows a plot of kinetics of the increase in absorbance versustime for PETINIA assays with representative data in Table 1.

FIG. 2 shows a standard curve for a PETINIA assay, derived from the datain Table 2.

FIG. 3 shows a correlation of the concentration data derived from anEMIT® assay to the concentration data derived from a PETINIA assay.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) Procainamide Analogs

Procainamide analogs which may be immobilized to a polymeric supporthave great potential in assays for procainamide in biological fluids.Varieties of such analogs have been prepared, and will be describedherein.

A first procainamide analog is 4-amino-N-(2-ethylamino-ethyl)benzamide,Formula 6:

This compound may be prepared from p-aminobenzoic acid, Formula 7, inaccordance with Scheme I. According to this procedure, the amino groupon p-aminobenzoic acid is protected by reacting the acid with a carbonicacid diester, giving an N-alkoxycarbonyl derivative of p-aminobenzoicacid. di-t-butyl dicarbonate is a particularly useful diester, producingthe protected aminobenzoic acid, Formula 8. The carbonyl group is thenactivated toward nucleophilic attack with N,N-disuccinimidyl carbonate,producing Formula 9, which may or may not be isolated. This compound isthen reacted with N-ethylethylenediamine to produce compound 10.Alternatively, protected compound 8 may be directly converted into anamide, Formula 10, by reaction with N-ethylethylenediamine in thepresence of dicyclohexyl carbodiimide (DCC). After preparation ofFormula 10, the protecting group may be removed by treatment with acid,generating Formula 6.

More broadly, this process may be used to synthesize a variety of amidesof Formula 11:

where R is hydrogen, alkyl having from 1 to 10 carbons, cycloalkyl, oran aromatic ring; and n is 1, 2, or 3. This is done by reacting Formula12:

with one equivalent of di-t-butyl dicarbonate for each amino group,producing a protected acid of Formula 1, where P is a t-butoxycarbonylgroup. The protected benzoic acid derivative is activated with DSC, andis then reacted with a polyamine of Formula 2:

where R is hydrogen, alkyl having from 1 to 10 carbon atoms, or aryl, toproduce a benzoic acid derivative of Formula 3. The t-butoxycarbonylprotecting groups may be removed by treating Formula 3 withtrifluoroacetic acid.

More elaborate procainamide analogs having long-chain substituents mayalso be prepared. The analogues of Formula 3 may be reacted with acompound of Formula 4:

where X is a leaving group; each CY₂ group is a methylene group, analkylmethylene group, or a carbonyl group; L is an alkylene group havingfrom 2 to 10 carbons or a group of the Formula (CH₂CH₂O)_(p)CH₂CH₂,where p is between 1 and 4; and P₂ is a protecting group. The result isan analog of Formula 5:

Particularly preferred compounds of Formula 4 include haloamides ofFormula 13:

Formula 13 may be obtained by reacting an co-bromoalkanoic acid (forexample, 5-bromopentanoic acid), Formula 14, with an activating agentsuch as N,N-disuccinimidyl carbonate to produce an activated carboxylicester. The activated ester is then reacted with a protected diamine toproduce a haloamide, Formula 15 (See Scheme 2).

Formula 10 is then reacted with Formula 15 to produce a protectedlong-chain benzamide, Formula 16.

Similarly, a protected diamine of Formula 17:

may be reacted with a protected acid ester of Formula 18:

to produce an amidoester of Formula 19:

Alternatively, Formula 18 may be activated by reacting the acid withN,N-disuccinimidyl carbonate, and then the activated compound may bereacted with Formula 17 to produce Formula 19.

The benzyl group on Formula 19 may then be removed by hydrogenolysis toproduce an amidoacid, Formula 20, which can then be reacted withprocainamide Formula 10 in the presence of dicyclohexylcarbodiimide orN,N-disuccinimidyl carbonate, giving a procainamide analog of Formula21:

The t-butoxycarbonyl protecting groups on the nitrogen atoms may then beremoved with trifluoroacetic acid to produce procainamide analog,Formula 22.

SYNTHESIS EXAMPLE 1 Preparation of Formula 22

(A) Preparation of Protected p-Aminobenzoic Acid

Protected p-aminobenzoic acid was prepared by dissolving 24 g (0.17 mol)p-amino-benzoic acid and 11.5 g (0.288 mol) sodium hydroxide in 300 mlwater. Di-t-butyl dicarbonate (42 g; 0.193 mol) was added to thesolution, but failed to dissolve, giving a nonhomogeneous mixture.Tetrahydrofuran (300 ml) and methanol (150 ml) were added to themixture, the solids dissolved and a homogeneous solution was obtained.The solution was stirred for 5.5 hours while monitoring pH, with 5.5 gNaOH added to the solution after the pH dropped to about 7.0. Theorganic solvents were removed from the reaction mixture with a rotaryevaporator, and the remaining aqueous solution was poured into 1000 mlwater and acidified with 75 ml 6 N HCl to a pH of between 3 and 4 withmechanical stirring. The white solid, which precipitated, was recoveredby filtration and dried in an oven under high vacuum. The dried solid,identified as the t-butyl carbamate, Formula 8, was obtained in a yieldof 28.11 g (68%).

(B) Activation of Protected p-Aminobenzoic Acid

The t-butyl carbamate of p-aminobenzoic acid (9.48 g; 40 mmol) andN,N′-disuccinimidyl carbonate (11.26 g; 44 mmol) were combined with 14ml triethylamine in 200 ml tetrahydrofuran (THF). The reaction mixturewas stirred under a nitrogen atmosphere at room temperature for threehours. Thin layer chromatography (TLC) was then performed using asolvent mixture of 50% hexane and 50% ethyl acetate to confirm that thereaction had reached completion. The reaction mixture was next added to100 ml of a saturated aqueous NaHCO₃ solution, and extracted three timeswith 250 ml ethyl acetate. The organic extracts were combined, and driedover anhydrous sodium sulfate. The solvent was removed in a rotaryevaporator, and the solid residue was dissolved in a mixture of 40 mlethyl acetate and 60 ml hexane. The solution was allowed to standovernight at room temperature, and a solid material recrystallized fromthe solution. This product, identified as activated ester, Formula 9,was isolated in a yield of 10.3 g (77%).

(C) Preparation of Formula 10

Activated ester, Formula 9 (1.077 g; 3.22 mmol), was dissolved in 6 mldichloromethane (DCM). The solution of Formula 9 was then addeddrop-wise to a room temperature solution of N-ethylethylenediamine(0.284 g; 3.22 mmol) in DCM under a nitrogen atmosphere. The reactionmixture was stirred for 5.5 hours. Dichloromethane (100 ml) was thenadded to the reaction mixture, and the resulting solution was extractedwith a saturated aqueous solution of sodium bicarbonate (20 ml). Theorganic phase was separated from the aqueous phase and dried over sodiumsulfate. The solvent was then evaporated, leaving a solid residueidentified as Formula 10.

(D) Preparation of Protected Procainamide Analog, Formula 21

Amidoacid, Formula 20 (520 mg; 1.494 mmol), N,N-disuccinimidyl carbonate(383 mg; 1.494 mmol), and 0.2 ml triethylamine were stirred at roomtemperature under a nitrogen atmosphere in a solution of 5 ml THF for 2hours. Benzamide, Formula 10, (458.6 mg; 1.494 mmol) was then addeddrop-wise to the THF solution, and the reaction mixture was allowed tostir overnight. The THF solvent was then evaporated under vacuum using arotary evaporator. Methylene chloride (100 ml) was then added to theresidue, and the resulting solution was washed twice with 10 mldeionized water. The organic phase was then dried over sodium sulfate,and the solvent was removed. The resulting crude product was purified bycolumn chromatography. The purified protected procainamide analog,Formula 21, was isolated in an amount of 570 mg (60%).

(E) Preparation of Procainamide Analog, Formula 22

Protected procainamide analog, Formula 21 (570 mg; 0.895 mmol), and 4 mlof a 40% solution of trifluoroacetic acid in dichloromethane werestirred at room temperature overnight. The solvent was then evaporatedfrom the reaction mixture. The residue was purified by columnchromatography using methanol as a diluent. A product identified asFormula 22 was isolated in a yield of 247.7 mg (64%).

(2) Use of Procainamide Analogs in Assays

The procainamide analogs of the current invention bind selectively toantibodies generated against procainamide. This allows the analogs to beused to indirectly or directly detect the presence of procainamide inbiological fluids. Thus, the present invention provides a powerful toolfor monitoring of procainamide levels.

The current invention may be used in a variety of test methods. Forexample, the inventive procainamide analogs may be immobilized onto atest strip, such as a microwell test strip, a glass slide or othersupport. One such method would involve derivativization of glass slideshaving 3-aminopropyl-triethoxysilane-coated glass slides.5-bromopentanoic acid is then activated with disuccinimidyl carbonate,and the amino-functionalized plated is allowed to react with thefunctionalized acid. The result is a plate having exposed bromoalkylgroups. This plate may then be reacted with the t-butyl carbamate ofp-amino-N-(ethylamino)ethyl-benzamide, Formula 6, in order to bindprocainamide analog, Formula 6, to the plate through a long-chainlinking group. A linking group is a portion of a structure whichconnects 2 or more substructures. A linking group has at least 1uninterrupted chain of atoms extending between the substructures. Theatoms of a linking group are themselves connected by chemical bonds. Thenumber of atoms in a linking group is determined by counting the atomsother than hydrogen. A linking group typically comprises about 2 toabout 15 atoms and may comprise a chain of from 2 to 8 atoms, eachindependently selected from the group normally consisting of carbon,oxygen, sulfur, nitrogen, halogen and phosphorous. Where the linkinggroup provides attachment of a protein to the hydroxyl group of thebenzene ring, the linking group usually comprises at least 5 atoms or,when less than 5 atoms, the linking group does not comprise solelycarbon atoms or oxygen atoms. For the most part, when a linking grouphas a non-oxocarbonyl group including nitrogen and sulfur analogs, aphosphate group, an amino group, alkylating agent such as halo ortosylalkyl, oxy (hydroxyl or the sulfur analog, mercapto) oxocarbonyl(e.g., aldehyde or ketone), or active olefin such as a vinyl sulfone orα-, β-unsaturated ester, these functionalities will be linked to aminegroups, carboxyl groups, active olefins, alkylating agents, e.g.,bromoacetyl. Where an amine and carboxylic acid or its nitrogenderivative or phosphoric acid is linked, amides, amidines andphosphoramides will be formed. Where mercaptan and activated olefin arelinked, thioethers will be formed. Where a mercaptan and an alkylatingagent are linked, thioethers will be formed. Where aldehyde and an amineare linked under reducing conditions, an alkylamine will be formed.Where a carboxylic acid or phosphate acid and an alcohol are linked,esters will be formed. Various linking agents are well known in the art;see, for example, Cautrecasas, J. Biol. Chem. (1970) 245:3059.

The plate having procainamide analog Formula 6 immobilized thereto maybe used within conventional immunoassays to detect the presence and/orconcentration of procainamide in a variety of assays.

The inventive analogs may also be used in Particle EnhancedTurbidometric Inhibition Immunoassays (PETINIA).

PETINIA is an assay format that utilizes latex particle-analyteconjugates, such as those described above, in order to determine analyteconcentrations in solution. PETINIA reactions are composed of threecomponents:

-   -   1) latex particles to which an antigen or an antigen analogue        has been attached;    -   2) antibodies generated to recognize the antigen; and    -   3) an assay buffer.

Non-specific agglutination (agglutination in the absence of antibody) ofthe latex-antigen conjugates is thought to be prevented due toelectrostatic repulsion between similarly-charged conjugates, and stericrepulsion of bound surface components (the antigens or antigenanalogues). Upon reaction of these groups with antibodies, the charge ofthe latex-bound moieties is altered, resulting in the formation ofinsoluble complexes. Accumulation of these complexes in the reactionsolution will increase the amount of light scattered when the reactionchamber is illuminated, the extent of which can be monitored byturbidimetry. The assay buffer used in this reaction must be optimizedto prevent non-specific reactions from occurring, which may affectparticle stability. The buffer is optimized in order to manage the shapeof the calibration curve and determine the concentration range ofanalyte detection.

When a sample containing free antigen (also referred to as an analyte)is present in the reaction solution, competition for the antibody occursbetween the bound antigen or antigen analogue in the particle conjugateand the analyte. This results in an inhibition of the formation ofinsoluble complexes and therefore an inhibition of the increase inturbidity. This inhibition can be calibrated by addition of knownconcentrations of analyte, enabling determination of analyteconcentration in various samples.

In most immunoassays, any cross-reactivity of metabolites of the analyteand any additional compounds that may be present in the samplepopulations should be understood, and if necessary, accounted for inorder to ensure that the result obtained is reflective of the trueanalyte concentration in the sample. For instance, it is good practiceafter addition of a sample to the solution containing the immobilizedantigen or antigen analogue, to account for any agglutination that mayhave been caused by the sample.

Exemplary latex polymers for use with this procedure include polystyrenelatex particles having functional groups that can react with aminogroups on their surface. Such functional groups include chlorosilyl;alkoxysilyl; activated carboxyl; chloromethylphenyl; epoxy groups; andCl—CY₂CH₂(CH₂)_(q)CY₂NH—L—NH— groups, where CY₂ is methylene or keto. Aparticularly useful polymer is a polystyrene latex polymer having apolystyrene core, with a glycidyl methacrylate polymer or copolymer as ashell. Procainamide analogs of Formula 3 or 8 may be directly reactedwith polystyrene/glycidyl methacrylate core/shell particles, binding theconjugates by reaction between the aliphatic amino group on the analogsand the reactive epoxide rings. Particles having styrene-butadiene latexcores and shells containing glycidyl methacrylate monomers orhydroxyalkyl methacrylate ester monomers are also suitable. Theprocainamide analogs of Formula 6 or 22 may be bound tostyrene-butadiene/glycidyl methacrylate core/shell particles in asimilar fashion. To bind the procainamide analogs tostyrene-butadiene/glycidyl methacrylate core/shell particles, theparticles may be reacted with N,N-disuccinimidyl carbonate, activatingthe exposed hydroxyl groups to SN₂ attack. The activated latex particlesthen are reacted with the procainamide analogs of Formula 6 or 22. Sincesteric repulsion between bound surface components plays a key role instability of the conjugate particles, it is preferred to conduct thecoupling reaction between the latex particle and the procainamide analogin the presence of a polyether polyamine linking agents, such as1,8-diamino-3,6-dioxaoctane (DA-10). This diamine tends to bind to anyunreacted glycidyl or activated hydroxyl groups on the surface of thelatex particles, increasing the level of bound surface components on theparticles without providing additional groups which will interact withthe procainamide antibody. The result is increased steric interactionbetween groups on the particle surface, resulting in greater particlestability.

Upon addition of antibody to assay medium with analyte containingsample, competition for the antibody occurs between the free analyte andthe particle-bound antigen. This competition results in an inhibition ofthe formation of insoluble complexes observed in the absence of analyte,and inhibits the increase in turbidity.

A description of a PETINIA based assay is as follows: A measure ofabsorbance r0 (λ=340-700 nm) in an empty sample cuvette is taken at timet0 (e.g., 0 sec.) and used as a control value. A known volume of aparticle reagent having a defined concentration of particle/procainamideanalog conjugate is added to the sample cuvette at time t1 (e.g., 16sec.), resulting in a solution having a known volume v0. An increasedabsorbance occurs upon addition of particle reagent. Generally 340 nm isthe wavelength of choice because the complex absorbs maximally at thatwavelength. At this point t2 (e.g., 66 sec.), a known volume of a samplesolution suspected of containing procainamide is added to the samplecuvette, increasing the solution volume to a volume v1. Absorbance r1 ata wavelength of 340-700 nm is then recorded at a time t3 (e.g., 102sec.), and a second absorbance r2 is recorded at a second, later, timet4 (e.g., 252 sec.). The absorbance at 700 nm is observed to subtractbackground noise. Certain other wavelengths as known to the skilled inthe art may also be used. The difference between these values at 340 nm(r2−r1) provides a good measure of the presence of nonspecific bindingbetween the sample and the particle reagent in the test solution; ifthis difference is greater than 50 mAU, the particle reagent may haveagglutinated in the absence of antibody, and the results of the test maybe unreliable.

A known quantity of antibody reagent is then added to the test solutionat time t5 (e.g., 282 sec.), increasing the solution volume to a volumev3. The absorbance undergoes an initial decrease due to the increase inthe solution volume; next, the absorbance undergoes a gradual increaseas the antibody reacts with the antigen particles, resulting in aformation of insoluble complexes. Any free antigen in the samplesolution will compete competitively with antigen particles for theavailable antibody, resulting in measurable inhibition of the increasein turbidity. Thus, the rate at which turbidity increases is dependenton the concentration of analyte present. At a later time, t6 (e.g., 438sec.), absorbance r3 is measured. Note that the specific time values andwavelengths provided here are exemplary only; a person skilled in theart will recognize that different values may also be used with success.

This absorbance data may then be used to obtain information about therate of particle agglutination. This rate is affected by competition forantibodies between the procainamide in solution and the particle-boundprocainamide analog. This competition leads to a reduction in the rateof particle agglutination. Under the assumption that changes in the rateof particle agglutination are dependent on the concentration of freeprocainamide in solution, agglutination rates may be used to derive theconcentration of free procainamide in solution. This rate data may becalculated using the following equation (Equation 1):Rate=r3−((v1/v3)*r2)−((1−v1/v3)*r0)  (Equation 1)In the above equation, (v1/v3) is the ratio of the solution volume atthe time absorbance measurement r1 is taken to the solution volume atthe time absorbance measurement r3 is taken. By analyzing several knownconcentrations of procainamide in solution, a standard curve can begenerated. From this curve, the rate calculated from the analysis of anunknown sample can then be directly translated into a procainamideconcentration via some typical curve fitting algorithm, such as a Logitfit.

The following precautions should be taken in using this method. If thedifference between r2 and r0 at 340 nm is greater than 1700 mAU, and/orthe difference between r2 and r0 at 700 nm is greater than 100 mAU, thereaction solution in the cuvette may contain foam, causing the resultsto be of dubious value. If r1−r0<350 mAU, or if r2−r1>50 mAU, there maybe particle agglutination in the absence of antibody (or non-specificagglutination). If the Final Optical Density (FOD) at 340 nm exceeds1700 mAU, the absorbance of the solution is above the linear responseregion and possibly the detection limit of the analyzer. The results ofthe PETINIA assay may then have unacceptable linearity, and should berejected.

Other examples of known immunoassay formats, which can be employed usingthe reporter reagents of the present invention, include directagglutination particle-based immunoassays, enzyme-linked immunosorbentimmunoassays, and fluorescence-based immunoassays.

SYNTHESIS EXAMPLE 2 Particle Reagent Preparation

(A) Preparation of Polystyrene/Polyglycidyl Methacrylate PolymerParticle

The polymer latex was an aqueous dispersion of polymer particles thatconsist of a core of poly(styrene) and a glycidyl methacrylate shell,prepared as described in Craig, U.S. Pat. No. 4,480,042.

(B) Conjugation of Procainamide to Polymer Particles

The procainamide-conjugated particle reagent was prepared using thefollowing reagents:

-   -   a) A solution of procainamide analog 22 was prepared by        dissolving 157 mg 22 in a mixture of DMSO and methanol to        produce a solution which contained 34.9 mg/ml 22 (80.2 mM).    -   b) A solution of DA-10 (1,8-diamino-3,6-dioxaoctane) was        prepared by dissolving 0.500 mL DA-10 in 10 mL water. Sufficient        aqueous HCl was then added to bring the pH of the solution to        9.2. The final solution had a DA-10 concentration of 39.34 mg/mL        (264 mM).    -   c) A wash buffer solution was prepared containing 0.464 g/L        sodium phosphate monobasic; 1.65 g/L sodium phosphate dibasic;        62.5 mL/L of a 16% aqueous solution of the surfactant Gafac;        0.06 g/L of neomycin sulfate; and 2.0 mL/L of the broad spectrum        antimicrobial Proclin 300. The pH of the solution was adjusted        to 7.8 by titration with 5N NaOH.

A polystyrene/polyglycidyl methacrylate particle solution, was used toprepare the particle reagent solution. 5 mL of a particle solutionhaving 10.3% solids was obtained. 0.243 mL of the solution ofprocainamide analog (80.2 mM stock solution), 0.10 mL of the DA-10solution (324 mM stock solution), and 0.27 mL of a 14.7% aqueoussolution of the surfactant Gafac were added dropwise with stirring tothe polystyrene/polyglycidyl methacrylate particle solution. Theresulting mixture contained concentrations of 3.50 mM procainamideanalog 22; 3.00 mM DA-10; and 0.07 wt. % Gafac. The final solids contentwas 9.24%. The pH was measured, and adjusted to 9.2. The reactionmixture was then allowed to react at 70° C. for 18 hr. At the end ofthis time, 10 mL of a wash buffer solution [1.0% Gafac, 15 mM Phosphate,0.2% Proclin 300 and 0.006% Neomycin Sulfate (pH 7.8)] was added to thereaction mixture, and the resulting mixture was subjected tocentrifugation at 28,000 rpm. The liquid supernatent was decanted, and20 mL wash buffer was added to the solid material. The solid materialwas resuspended in the wash buffer by sonication. The process ofcentrifugation and resuspension was repeated 3 more times. After thefinal centrifugation, the final solid pellet is taken up into sufficientwash buffer to have a final volume of 20 mL, and a final particleconcentration of 50 mg/mL.

IMMUNOASSAY EXAMPLE 1 Use of Particle Reagent in PETINIA Assays forProcainamide

A. Calculation of Particle Agglutination Rates.

The procainamide-particle conjugates prepared as described above werediluted to a concentration of 2.0 mg/mL in the wash buffer solution toobtain a particle reagent (PR). Anti-procainamide monoclonal antibodieswere diluted in antibody diluent to prepare an antibody reagent (AB).The antibody diluent had a pH of 6.5, and included 100 mM Phosphate, 1 MNaCl, 1% BSA, 0.2% Proclin 300 and 0.006% Neomycin Sulfate. Theconcentration of antibodies in the diluent was 75 μg/mL. An assay bufferhaving a pH of 7.7 comprising phosphate (100 mM); 0.3 wt. % Gafac; KCl(100 mM); 0.2 wt. % Proclin 300; and 0.006 wt. % neomycin sulfate wasprepared.

The assay was carried out on a Dimension® Clinical Chemistry system fromDade Behring Inc, Deerfield, Ill., using a sample solution containingfree procainamide. At time t=0 sec., the absorbance r0 of an emptycuvette at 340-700 nm was measured as a control air blank. At time t=16sec., 80 μl PR and 130 μl of assay buffer followed by 103 μl of waterwere added to the cuvette. At time t=66 sec, 2 μl of sample followed by32 μl of water were added to the cuvette. Later, at time t=102.9 sec.,the absorbance r1 of the test solution was recorded at a total solutionvolume v1 of 347 μl. A second absorbance r2 was recorded at time t=254.4sec. The difference between r2 and r1 was found to be less than 50 mAU;therefore, the risk of particle agglutination from excessivenon-specific binding was deemed to be acceptable. Thus, at time t=284.8sec., 80 μl of AR followed by 40 μl of water was added to the testsolution. Finally, at time t=436.3 sec, the absorbance r3 of the testsolution was recorded at a final solution volume v3 of 347 μl. Theabsorbance of the test solution as a function of time has been plottedin FIG. 1; and the numerical results of this experiment have beentabulated in Table 1. Using the numbers generated from the experimentdescribed above, a measurement of the rate of particle agglutination canbe derived from the above results, in accordance with equation 1. Usingthis expression, a Rate value of 176.0 mAU may be calculated.

TABLE 1 Absorbance as a Function of Time During a PETINIA Assay.Absorbance (mAU) Time (Sec.) Volume (μL) (340-700 nm) 0  0  65.73 (r0)18.1 313 929.92 66.6 347 950.04 102.9 347 (v1) 871.74 (r1) 254.4 347898.04 (r2) 284.8 467 902.43 436.3 467 (v3) 860.09 (r3)

B. Determination of a Standard Curve For PETINIA Assays.

The procedure described above for determination of Rate values wasapplied to sample solutions having known procainamide concentrations of0 μg/mL, 2.16 μg/mL, 4.33 μg/mL, 8.66 μg/mL, and 17.32 μg/mL. For eachsolution, values correlating to the rate of particle agglutination werecalculated in accordance with Equation 1, and the calculated data wastabulated in Table 2. The rate data from Table 2 was fit to a logitcurve in order to generate a standard curve, shown in FIG. 2.

TABLE 2 Rate of Agglutination as a Function of Free ProcainamideConcentration. [PROC], μg/mL 0 2.16 4.33 8.66 17.32 Rate 224.07 178.86129.70 55.47 24.21 (mAU)

C. Comparison of Results from PETINIA Assay and EMIT® Assay.

The effectiveness of the PETINIA assay as described above was verifiedby comparing the results of PETINIA assays to the commonly used EMIT®assay (Enzyme Multiplied Immunoassay). The EMIT® assay was performed onan aca® discrete clinical analyzer from Dade Behring Inc., Deerfield,Ill, using purified water and an aca® analyzer PROC Analytical TestPack. The analyzer performed the test steps automatically at atemperature of 37° C. In brief, a 40 μL sample of human serum wasdiluted with purified water by the analyzer. Tablets containing 500 μmolof TRIS buffer and 0.9 mmol of NaCl from the Analytical Test Pack wereadded to the test solution. Then, 66 μL of a solution of sheep-derivedprocainamide antibody was added to the sample solution from theAnalytical Test Pack by the analyzer. 66 μL of a solution ofprocainamide/glucose-6-phosphate dehydrogenase conjugate was next addedto the sample solution, and allowed to complex to any antibody that hadnot already complexed to procainamide in the original serum sample.Next, a tablet containing the enzyme substrate glucose-6-phosphate (75μmol) and NAD⁺ (65 μmol), and allowed to react with any non-complexedconjugate, forming NADH and 6-phosphogluconolactone. The analyzermonitors the change in absorbance at 340 nm to determine the increase inNADH concentration, and derives the original procainamide concentrationfrom this value.

A number of samples believed to contain procainamide were then tested bythe above-described EMIT® method, and the data are recorded in Table 4.The same set of samples was then retested, using the PETINIA assaytechnique described in Example 1 and recorded in Table 4. To determinehow well the values derived from the two methods correlate, theconcentrations from the EMIT® data from the aca® analyzer were plottedagainst the concentrations from the PETINIA data (FIG. 3). The resultwas a straight line having a slope of 1.03, and a y-intercept of 0.64.The R squared value was 0.9944, the ideal value of 1.00. These resultsdemonstrate that the two methods produce results that correlate well.

TABLE 4 Comparison of Results from a PETINIA Assay to Results from anEMIT ® Assay. PETINIA EMIT ® Analyte Analyte Absorbance ConcentrationConcentration Sample # (mAU) (μg/mL) (μg/mL) #1 162.42 2.91 2.5 #2147.62 3.49 2.7 #3 128.27 4.28 3.4 #4 111.37 5.04 4.4 #5 139.05 3.84 2.9#6 155.12 3.20 2.5 #7 104.70 5.36 4.3 #8 72.08 7.34 6.4 #9 21.85 18.7217.5 #10 137.68 3.89 3.2 #11 178.52 2.27 1.5 #12 168.48 2.67 1.8 #1390.00 6.15 5.6 #14 124.84 4.43 3.9 #15 125.49 4.40 3.7 #16 213.76 0.620.1 #17 113.33 4.95 4.5 #18 71.86 7.36 6.4 #19 119.70 4.66 3.8 #20181.74 2.14 1.2 #21 196.76 1.49 0.5 #22 97.98 5.71 4.6 #23 112.47 4.993.8 #24 142.48 3.70 2.9 #25 183.94 2.05 1.2 #26 119.52 4.66 3.8 #27101.07 5.54 4.9 #28 161.68 2.94 1.9 #29 117.39 4.76 3.5 #30 176.48 2.351.6 #31 135.20 3.99 3.4 #32 102.65 5.46 4.9 #33 138.55 3.86 2.9 #3454.42 8.98 7.9 #35 54.02 9.03 7.8 #36 115.75 4.83 4.7 #37 65.98 7.84 7.2#38 32.91 12.83 11.7 #39 31.51 13.26 11.9 #40 48.11 9.78 8.7 #41 90.376.13 5.6 #42 34.35 12.42 10.6 #43 54.30 8.99 8.3 #44 118.75 4.70 4.1 #4576.30 7.03 6.5 #46 32.40 12.98 11.9 #47 41.84 10.78 9.8 #48 61.98 8.207.6 #49 90.07 6.15 5.8 #50 32.78 12.86 11.8 #51 99.77 5.61 5.1 #52 47.269.90 8.9 #53 64.81 7.94 7.3

Although the present invention has been described in detail withparticular reference to preferred embodiments thereof, it should beunderstood that the invention is capable of other different embodiments,and its details are capable of modifications in various obviousrespects. As is readily apparent to those skilled in the art, variationsand modifications can be affected while remaining within the spirit andscope of the invention. Accordingly, the foregoing disclosure,description, and figures are for illustrative purposes only, and do notin any way limit the invention, which is defined only by the claims.

1. A latex polymer-bound procainamide analog, having a structurerepresented by of: a compound of Formula 7:

wherein: R is hydrogen or alkyl; each CY₂ group is a carbonyl group; Lis a linking group selected from the group consisting of (CH₂)_(m) and(CH₂CH₂O)_(p)CH₂CH₂, where m is between 2 and 10 and p is between 1 and4; polymer is a polymer having functional groups that react withaliphatic amino groups; n is 1; and q is between 2 and
 10. 2. The latexpolymer-bound procainamide analog of claim 1, wherein the polymer is inthe form of particles.